Method for separating charged biologically active substances from liquids and the recovery thereof

ABSTRACT

The present invention relates to a method for the at least temporary retention of charged biologically active substances such as endotoxins, viruses, and proteins from liquids, and optional later release for better determination. The object is achieved by a method for the at least temporary separation and/or detection of charged biologically active substances in a liquid by means of electrosorption and/or electrofiltration, comprising the following steps: a. providing a polymer membrane with a flat and porous metal coating at least on a first side of the polymer membrane; b. providing a counterelectrode; c. applying a voltage between the metal coating of the polymer membrane and the counterelectrode; d. bringing the polymer membrane and the counterelectrode into contact with the liquid, with the contacting being performed such that the liquid generates at least one connection between the polymer membrane and the counterelectrode.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.16/473,640 filed Jun. 26, 2019, which is a National Stage Entry ofPCT/EP2017/084721 filed Dec. 28, 2017, which claims priority from GermanPatent Application Serial No. 102016125818, filed Dec. 28, 2016, theentire disclosures of which are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a method for the at least temporaryretention of charged biologically active substances such as endotoxins,viruses, and proteins from liquids, and optional later release forbetter determination.

BACKGROUND Background Information

Endotoxins are lipopolysaccharides (LPS) and components of the outermembrane of the cell wall of Gram-negative bacteria. They consist of alipophilic lipid that is anchored in the membrane and a hydrophilicpolysaccharide, which represents the antigenic properties. Endotoxinsare released upon lysis of the bacterial cell. They are extremelyheat-stable and can even be detected after sterilization—that is, afterthe bacteria have been killed. Because of their ability to induce animmune response, endotoxins are among the pyrogens. Endotoxins in aquantity of 1 ng/kg body weight are considered sufficient to cause afebrile response (ARDUINO (1989)). Besides fever as a result ofinflammation, they can cause numerous physiological reactions in humansafter contact with mucous membranes and especially upon passing into thebloodstream. These include hypotension, blood coagulation, andcomplement activation, as well as life-threatening states of shock.

Endotoxins are usually detected in rabbits or by means of a Limulusamebocyte lysate (LAL) test. The LAL test is based on the blood of thehorseshoe crab, which reacts extremely sensitively to endotoxins ofGram-negative bacteria. Due to its sensitivity, the LAL test is the mostwidely used test in the fields of pharmacology and medicine. However,endotoxins can only be determined in clear, undyed fluids when usingthis test. Direct determination of endotoxins in human blood istherefore not possible. It is for this reason that the so-calledmonocyte activation test was developed in recent years. This works byusing different levels of enzymatic reactions that mimic the human feverresponse. It also requires the creation of a standard curve for eachdetermination, since the reactants of each test kit react differently.This test requires special training, has high specific costs, and alsotakes much longer than the LAL test.

Microporous membranes have long since been known. These are madeprimarily of polymers and used for water treatment (wastewater, drinkingwater, industrial waters) as well as in the pharmaceutical industry forthe production of ultrapure water and in medical technology as sterilefilters or respiratory filters. The areas of application are numerousand very divergent. Microporous membranes typically have a pore sizebetween 0.01 μm and 10 μm and retain substances according to these poresizes.

Microporous filters are typically used to separate substances that aredissolved in water and to obtain a clear filtrate. This is generallydone mechanically through the pore size. All substances that are largerthan the size of the pores are mechanically retained. In addition tothis property, there is another mechanism that takes place in order toretain substances when they pass through the membrane. This is anundefined adsorption by the materials making up the membrane itself,such as polyether sulfone, polypropylene, or polyvinylidene fluoride(PVDF), for example. Different materials adsorb different solutes todifferent degrees (“Analyte loss due to membrane filter adsorption asdetermined by high-performance liquid chromatography,” M. Carlson and R.D. Thompson, Journal of Chromatographic Science, Vol. 38, February2000).

Targeted adsorption of substances smaller than the pore size of themicroporous membrane by means of the material properties of the membranematerial is achieved through treatment of the chemical composition ofthe membrane material. A positive charge is generated, for example, bycombining the membrane material with positively charged quaternaryammonium compounds. Positively charged membranes are known from U.S.Pat. No. 5,282,971 or from U.S. Pat. No. 7,396,465 B2, while negativelycharged membranes are known from U.S. Pat. No. 7,132,049 B2. Forexample, positively charged microporous membranes are used tomechanically retain bacteria and allow positively charged material topass through in order to avoid nontargeted, unquantifiable adsorption bythe membrane material. However, positively and negatively chargedmembranes are also used to bind and concentrate proteins by adsorption.Positively charged microporous membranes are also used to bindendotoxins and viruses through adsorption in addition to filtration, asis disclosed in DE 1999981099947 A1, for example.

CH 678403 discloses a metal-coated membrane with optionally slightlyporous passages between macropores on one side and micropores on themetallic side. In addition, metal membranes with tunnel-like passagesare known from DE 101 64 214 A1, for example. These differ from passagesthat are porous in the parlance of the application, such as those whichare known from porous polymer membranes, for example, in that they formno cavities outside of the actual passage channel within the membrane.Therefore, “porous” is not to be equated with the characterization thatthe membrane has pores, i.e., passages, like in DE 101 64 214 A1, forexample. Porous passages thus have a surface area within the membranethat substantially exceeds the surface area of a circular tunnel withthe same pore size through a membrane of equal thickness, at least by50%, particularly by a multiple, more particularly by at leastthreefold.

Moreover, it is known from WO 1999/22843 A1 to sputter a polymermembrane with metal.

It is also known from U.S. Pat. No. 4,857,080 to seal a membrane with ametal coating.

Another form of adsorption is electrosorption. Electrosorption isaccomplished by forming an electrically charged field on surfaces byapplying a positive and negative voltage to two electrodes. Acombination of electrosorption and ultraporous filtration is describedin “Removal of arsenic and humic substances (HSs) byelectro-ultrafiltration (EUF)” (Weng, Y.-H. et al., Chem. Eng. R&D Vol.77, July 1999, pages 461-468). In that case, an increase of 30% to 90%in the adsorption of negatively charged arsenic (V) during thefiltration of arsenic-contaminated water is achieved by ultrafiltrationby establishing an electric field by means of an external electrode thatis positioned near an ultrafiltration membrane. A similar use ofelectrosorption in combination with membranes is described in US2013/0240361 A1. The purification of dialysis water is described in acombination of substances with highly adsorptive properties and theregeneration thereof by electrical charging. The method is carried outin conjunction with a dialysis membrane filter.

An electrosorption membrane is described in EP 0872278 A1. Here, aceramic membrane is provided with a conductive layer of pyrolyticcarbon. The pores are sealed with pyrolytic carbon, and the ceramicsurface is then rendered conductive through high-temperature conversionof the ceramic surface to carbide. With this ceramic membrane, saltswere adsorptively bound on the surface by electrosorption. Possibleelectrosorption on the conductive surface of a ceramic membrane allowsfor a more flexible sorption of substances but is very expensive toachieve. The pores of the membrane are sealed during the process forproducing the conductive surface, and the ceramic surface is providedwith a conductive carbide layer in a subsequent production step by meansof very high temperatures.

In addition to the advantages of membranes that have been positively ornegatively charged by chemical treatment—namely the combination ofmechanical filtration and adsorption—they also have a drawback. Sincethe charge cannot be varied, the substances that have been adsorptivelybound can be removed again or recovered once the membrane has beenloaded only by shifting the charge through a solution to be filtered,generally by means of a change in pH. In particular, this represents anadditional expense during the recovery of active substances such asproteins through concentration. Endotoxins are denatured by shifting thepH into the basic range. But this pH shift would be necessary in orderto recover endotoxins from a positively charged membrane. It istherefore not possible to obtain endotoxins—from blood, for example—bymeans of permanently charged membranes in order to subsequently convertthem into a colorless liquid and forward them for easier analysis.

SUMMARY

The object is to establish a method for the at least temporaryseparation and/or detection of charged biologically active substances,particularly endotoxins, from liquids, particularly from coloredliquids.

The object is also to establish a method for the at least temporaryretention of charged biologically active substances, particularlyendotoxins, in order, for example, to enable the content of endotoxinsto be determined independently of the other substances of the originalfluid.

It is also the object to provide a corresponding device.

The objects are achieved by a method for the at least temporaryseparation and/or detection of charged biologically active substances ina liquid by means of electrosorption and/or electrofiltration,comprising the steps of a. providing a polymer membrane with a flat andporous metal coating at least on a first side of the polymer membrane;b. providing a counterelectrode; c. applying a voltage between the metalcoating of the polymer membrane and the counterelectrode; and d.bringing the polymer membrane and the counterelectrode into contact withthe liquid, with the contacting being performed such that the liquidgenerates at least one connection between the polymer membrane and thecounterelectrode. The objects are further achieved by anelectrofiltration and/or electrosorption device comprising at least onepolymer membrane with a flat and porous metal coating on at least oneside of the polymer membrane and a contact of the metal coating and acounterelectrode and, in particular, an additional electrode, whereinthe polymer membrane and the counterelectrode are arranged in a housingthat is embodied particularly as a syringe attachment and/or has a smallhold-up volume, particularly of no more than 10 ml and/or no more than20 mm³/mm² of metal coating on the polymer membrane, particularly nomore than 2 mm³/mm² of metal coating on the polymer membrane, and/orwherein the polymer membrane and the counterelectrode are connected to avoltage source that is configured to form a voltage between polymermembrane and counterelectrode, wherein the voltage source with thepolymer membrane and the counterelectrode are arranged particularly in acommon housing, and/or a current-measuring device is provided thatmeasures the current flowing between polymer membrane andcounterelectrode and/or the rate of change thereof and/or compares itwith a limit value, and/or the voltage source is configured to reverseand/or reduce the polarity of the voltage.

Advantageous developments include a method wherein step c takes placeafter steps a and b and before step d, and/or wherein step d takes placeafter steps a and b and before step c. Further advantageous developmentsinclude a method wherein, after steps a to d, the liquid is removed atleast partially and/or the liquid is allowed to pass through themembrane at least partially. The method may further include that aftersteps a to d, a polarity reversal and/or reduction of the voltage takesplace, with rinsing of the membrane being performed particularly beforeand/or after the polarity reversal and/or reduction. The polymermembrane with a flat and porous metal coating and the counterelectrodeare accommodated in a housing, which, in particular, has a small hold-upvolume, particularly of no more than 10 ml and/or no more than 20mm³/mm² of metal coating on the polymer membrane, particularly no morethan 2 mm³/mm² of metal coating on the polymer membrane, and the liquidto be filtered is conducted through the housing. In the method, theliquid is pressed out of a syringe against the polymer membrane with aflat and porous metal coating or through the polymer membrane with aflat and porous metal coating and/or through the housing throughactuation of the syringe. The counterelectrode is formed either by anadditional flat, porous metal coating on a second side that is situatedopposite the first side, the two-dimensional metal coatings beingisolated from each other by the polymer membrane, or through arrangementof a permeable electrode that is formed particularly by a metallic meshwith interposition of an insulating and permeable spacer. The method ischaracterized in that the porosity of the polymer membrane with metalcoating is reduced by between 1% and 50%, in particular 1 and 20%relative to the initial bubble point pore and/or the mean pore sizecompared to the uncoated polymer membrane. The method is furthercharacterized in that the thickness of the metal coating is from 5 to 50nm and the pore size of the uncoated polymer membrane is particularlygreater than 0.01 μm. The method is used for determining the occupancyof the binding sites of the polymer membrane with a flat and porousmetal coating and/or for determining at least one concentration in theliquid, wherein the current flow caused by the applied voltage isdetected and/or evaluated, particularly evaluated with regard to fallingbelow a limit and/or exceeding a positive and/negative rate of change,and an alarm is triggered particularly in the case of an undershoot orovershoot.

Further advantageous developments in the electrosorption and/orelectrofiltration device include that the counterelectrode is eitherformed by an additional flat, porous metal coating on a second side thatis situated opposite the first side or by a permeable electrode that isformed particularly by a metallic mesh with interposition of aninsulating and permeable spacer. In the electrosorption and/orelectrofiltration device, the porosity of the polymer membrane withmetal coating is reduced by between 1% and 50%, in particular 1 and 20%relative to the initial bubble point pore and/or the mean pore sizecompared to the uncoated polymer membrane. Furthermore, in theelectrosorption and/or electrofiltration device, the thickness of themetal coating is from 5 to 50 nm and the pore size of the uncoatedpolymer membrane is particularly from 0.01 μm to 15 μm.

The production of a polymer membrane with a flat and porous metalcoating for use in the method and/or the device can be achieved throughdeposition by means of magnetron sputtering. This allows for thelarge-scale production of thin layers with a homogeneous layer thicknessas well as a complex layered structure. The basis of the magnetrondeposition is a plasma discharge in an inert gas atmosphere, e.g.,argon, that is amplified by a static magnetic field (A. Anders, Handbookof Plasma Immersion Ion Implantation and Deposition, Wiley-VHC, 2004).The ions of the process gas are accelerated cathode, knocking atoms outof the cathode on impact. Consequently, the cathode (target) must bemade of the material that is to be deposited. The atoms knocked out ofthe target then condense on the substrate to be coated and form acontinuous thin layer. This layer thickness can be produced in acontrolled manner so as to be from few nanometers to severalmicrometers. Besides round magnetrons, rectangular variants that areseveral meters in length are widely used above all for coating largesurfaces, for example in architectural glass coating. Surfaces ofmembranes can be coated in this way.

In order to produce the polymer membrane with a flat and porous metalcoating, a polymer membrane (for example, polysulfone, polyethersulfone,polypropylene, or polyvinylidene fluoride) can be provided with a thinlayer of metal by means of the magnetron sputtering process. Theresidence time of the membrane in the process is selected so as to beshort enough that the temperature remains below 200° C., particularlybelow 100° C., and the original chemical structure of the polymermembrane is not affected. As an example, a polyethersulfone membranehaving a microporous structure was provided with a 20 nm-thin layer ofaluminum. Porosity analyses were performed on this membrane. Thefollowing table shows the results of the porosity measurement of themembrane in the original state on the one hand and with a layer ofaluminum with a defined thickness of 20 nm on the other hand.

TABLE 1 Pore size of a microporous polyethersulfone membrane in theoriginal and with a 20 nm-thick layer of aluminum. Polyethersulfonemembrane Difference 20 nm coating Al Original % Bubble point pore size(μm) 0.51 0.56 8.5 Mean pore size (μm) 0.41 0.43 6.1 Smallest pore size(μm) 0.37 0.39 5.5

It can be seen that the porosity of the membrane is influenced to below10%.

The metal coating is applied at least two-dimensionally to a first sideof the polymer membrane and/or at least to the surfaces that can beaccessed from one side, particularly until the layer thickness of themetal coating of the polymer membrane is between 1% and 45% relative tothe initial bubble point pore and/or the mean pore size of the uncoatedpolymer membrane.

Particularly with a preferred coating up to a thickness of up to 200 nm,it is possible to hardly influence the pore size of the polymermembrane, in particular the polysulfone membrane, particularly in therange from 0.1 μm to 10 μm, and to thus leave it largely unchanged.

The coating is particularly porous and, in particular, applied directly.

Copper, aluminum, silver, gold, nickel, platinum, and/or tungsten oralloys containing copper, aluminum, silver, gold, nickel, platinum,and/or tungsten can be used as the metal for the coating.

Membranes of polysulfone, polypropylene, polyethersulfone,polyetherimide, polyacrylonitrile, polycarbonate, polyethyleneterephthalate, polyvinylidene fluoride (PVDF), and/orpolytetrafluoroethylene or membranes containing polysulfone,polypropylene, polyethersulfone, polyetherimide, polyacrylonitrile,cellulose, polycarbonate, polyethylene terephthalate, polyvinylidenefluoride (PVDF), and/or polytetrafluoroethylene can be used as thepolymer membrane, for example.

It is with particular advantage that metal is deposited until the layerthickness of the metal coating of the polymer membrane is between 1% and45% relative to the initial bubble point pore and/or the mean pore sizeof the uncoated polymer membrane.

These values enable good conductivity to be combined with goodthroughput and high porosity.

Preferably, metal is deposited until the porosity of the polymermembrane with metal coating is reduced by between 1% and 50%, inparticular 1 and 20% relative to the initial bubble point pore and/orthe mean pore size compared to the uncoated polymer membrane. Thesevalues also enable good conductivity to be combined with good throughputand high porosity.

Preferably, metal is deposited until the initial bubble point poreand/or the mean pore size of the polymer membrane with metal coatingand/or aluminum oxide is 0.01 to 10 μm. For this purpose, the polymermembrane is selected so as to have an initial bubble point pore and/ormean pore size of greater than 0.01 to 10 μm.

Advantageously, the polymer membrane is coated on the first side and ona second side that is situated opposite the first side porously anddirectly with metal. In particular, the two-dimensional coatings,including metallizations, are electrically insulated from one another.

Through omission of the coating of the porous passages and of the edgesto the extent that no conductive connection is formed between the twosides, a membrane with two electrically conductive and mutuallyinsulated surfaces can be produced.

Advantageously, metal has been deposited until the thickness of themetal coating or the mean thickness of the metal coating is at least 1nm, particularly at least 5 nm, and no more than 50 nm. These values ofat least 5 nm enable good conductivity to be combined with goodthroughput and high porosity.

Advantageously, the pore size of the uncoated polymer membrane isselected so as to be between 0.01 and 15 μm, particularly up to 10 μm,and/or greater than or equal to 0.1 μm. This enables a sealing of thepores with metal to be prevented particularly well.

Advantageously, metal has been deposited until the thickness of themetal coating of the pores within the membrane or the mean thickness ofthe metal coating of the pores within the membrane is at least 1 nm andno more than 50 nm.

The object is also achieved through the use of a metal-coated polymermembrane having porous passageways, the metal-coated polymer membranehaving an internal polymer membrane with porous passages and a metalcoating, characterized in that the polymer membrane is completelyencapsulated by the metal coating and the metal coating has a thicknessof from 1 nm, particularly 5 nm, to 500 nm.

In particular, the coating is applied directly to the polymer membrane.In particular, the coated polymer membrane consists exclusively of thepolymer membrane and the metal coating.

To special advantage, the polymer membrane with metal coating thus has aporosity that is reduced by between 1% and 50%, particularly 1% and 20%relative to the initial bubble point pore and/or the mean pore sizecompared to the uncoated polymer membrane.

For example, the membrane can also be folded and/or used in folded formas is known in conventional membranes. In particular, at least oneinsulating folding aid is used and/or included, particularly on eachside of the membrane. These allow for passage of liquid and, inparticular, provide for insulation of the individual folds from oneanother.

In particular, the at least one folding aid is arranged on one or bothsides of the membrane prior to folding and folded together with themembrane. The folding aids need not be made completely of insulatingmaterial; for instance, a polymer fleece can be used that isparticularly coated in an electrically conductive manner on one side orboth sides, but the fleece itself provides for insulation.

According to the method, in order to achieve the object of the at leasttemporary separation and/or detection of charged biologically activesubstances,

-   -   a. a polymer membrane with a flat and porous metal coating,        particularly as described above, is provided at least on a first        side, particularly on both sides, of the polymer membrane;    -   b. a counterelectrode is provided;    -   c. a voltage is applied between the metal coating of the polymer        membrane and the counterelectrode;    -   d. the polymer membrane and, in particular, the counterelectrode        are brought into contact with the liquid, with the contacting        being performed particularly such that the liquid generates at        least one connection between the polymer membrane and the        counterelectrode.

By means of such a method, it is possible to adsorb and/or retain, andhence particularly to separate charged substances, particularlybiologically active charged substances, as well as other chargedsubstances at least temporarily from liquids—for example fromblood—through binding to the polymer membrane with metal coating. At thesame time, a much more customized and flexible process control can beachieved than is possible with known (ionically) charged membranes.Detachment into another liquid, for example—for the purpose of recovery,for example—can also be facilitated, particularly without the need tochange a pH value, by reversing the voltage, which does not presupposethe same level of voltage with an reversed sign, but rather can also beof a lower or higher magnitude, and/or by decreasing the voltage.

The amount of voltage is advantageously no more than 1.5 V. Inparticular, the amount of voltage is no more than 1.5 V even after apolarity reversal. The voltage or the energy required to generate it canbe transferred capacitively, inductively, and/or by cable, for example.In particular, the voltage or the energy required to generate it can betransferred, especially inductively, into a housing that encloses thepolymer membrane and the counterelectrode. For example, the voltage orthe energy required to generate it can be inductively coupled into thehousing and applied to the polymer membrane and the counterelectrodewithin the housing via cables.

The expression “charged biologically active substances” is to beunderstood as referring to a variety of substances. A more or lessspecific retention, separation, and/or a more or less specific detectionof individual or numerous substances can be made possible, for example,through targeted selection of the voltage and/or of the membranesurface. A retention and/or an adsorption against/by the membrane ofmore highly charged particles is already possible at lower voltages thanfor less-highly charged particles. This allows for a certain selectivitythrough selection of the voltage. It is not imperative that only asingle substance be separated and/or detected. Examples of suitablebiologically active substances include viruses, bacteria, endotoxins,proteins, amino acids, zwitterions, substances with an isoelectricpoint, exosomes, and/or vesicles. They need not have a charge in everyenvironment in order to be regarded as charged. It is sufficient thatthey have a charge in a state in which they are or can be brought intocontact with the membrane; in particular, they are present in the liquidat least partially in a charged state. Thus, separation and/ordetection, particularly of proteins, more particularly viruses, isperformed particularly in a solution having a pH at which the substancesto be retained and/or adsorbed have the greatest possible charge,particularly relative to substances that are not to be retained oradsorbed.

In general, polymer membrane with metal coating and, in particular, thecounterelectrode as well are extensively brought into contact with theliquid, particularly wetted over a large area. A contact or connectionover a small area—by means of a drop between the counterelectrode andpolymer membrane with metal coating, for example—is already sufficient,however. Contacting can also be achieved by filling one or a pluralityof pores of a two-sided metallically coated polymer membrane, one sideof which is used as a counterelectrode.

To special advantage, the counterelectrode is formed either by anadditional flat, porous metal coating on a second side that is situatedopposite the first side, the two-dimensional metal coatings beingisolated from each other by the polymer membrane, or through arrangementof a permeable electrode that is formed particularly by a metallic meshand/or a rod electrode with interposition of an insulating and permeablespacer and/or with spacing.

Advantageously, the porosity of the polymer membrane with metal coatingis selected so as to be reduced by between 1% and 50%, in particular 1and 20% relative to the initial bubble point pore and/or the mean poresize compared to the uncoated polymer membrane. This provides reliableconductivity and large porosity at the same time.

Advantageously, the polymer membrane is selected such that the thicknessof the metal coating is 1 nm, particularly 5 to 50 nm, and/or the poresize of the uncoated polymer membrane is particularly greater than 0.01μm and particularly less than 15 μm. This provides reliable conductivityand large porosity at the same time.

To special advantage, a reference electrode is provided for measurementpurposes and the potential at the reference electrode is measured.

Advantageously, in addition to the polymer membrane with metal coatingthat is used according to the invention as an electrode andcounterelectrode and, optionally, as a reference electrode, at least oneadditional electrode is provided, particularly at least one additionalpolymer membrane with a metal coating or an additional side of thepolymer membrane with a metal coating, particularly as explained above,provided as an additional electrode. In particular, this additionalelectrode is likewise arranged in the common housing and is insulatedelectrically from the polymer membrane with metal coating and thecounterelectrode. The common housing can also be an outer housing thatencloses an inner housing in which polymer membrane and counterelectrodeare arranged.

In particular, a voltage is applied to the at least one additionalelectrode that is selected such that the potential of thecounterelectrode comes to lie between the potential of the polymermembrane with metal coating and of the at least one additionalelectrode. In particular, the counterelectrode is arranged in thehousing between the polymer membrane with metal coating and the at leastone additional electrode.

In this case, it can also be expedient to use a plurality of referenceelectrodes and to respectively arrange them between the electrodesand/or counterelectrode.

Electrosorption experiments were conducted on a polyethersulfonemembrane with a pore size of 0.2 μm in an arrangement as shown inFIG. 1. A laboratory filtration membrane with a diameter of d=47 mm wasprovided for this purpose with a 15 nm layer of aluminum by means ofmagnetron sputtering. A copper cable was glued to the aluminum surfaceand provided with an insulating varnish. The remaining cable was about30 cm long and insulated. The membrane was placed in a commercial vacuumfiltration unit. The receiving vessel was filled with pure water and aplatinum counterelectrode introduced therein.

Endotoxins were introduced into the pure water in the receiving vesselso that an endotoxin concentration of 1,000 IU (international unitsendotoxin) was achieved. Filtration was carried out without pressure.Filtration was performed with the membrane in the original state withouta coating and with a membrane with a 15 nm coating. A voltage of 500 mVwas applied to the coated membrane. The results are shown in thefollowing table.

Concentration of ml endotoxin in IE/ml Sample feed collecting vesselvolume Filter without coating 1000 952.5 100 Filter with coating at +500mV 1000 0.3 50

It can clearly be seen that endotoxins are retained, particularlyadsorbed almost completely via the charged aluminum coating, so that theconcentration in the collecting vessel is close to zero. Little wasadsorbed not only the polymer membrane in the original state withoutmetal coating, but also the metal-coated polymer membrane withoutvoltage.

The adsorbed endotoxins can be released again and removed from themembrane by reversing the polarity of the voltage and rinsing themembrane.

Similar to the endotoxin retention experiment described above, virusretention can also be achieved. It is known that viruses have a negativecharge above their isoelectric point and can be adsorbed on surfaceswith a positive charge (Adsorption of viruses to charged modifiedsilica, Zerda et al., Applied and Environmental Microbiology, January1985, p. 91-. Using the same experimental setup as described above inrelation to the retention of bacteria, experiments were conducted on theretention of viruses. Bacteriophages MS2 with a size (diameter) of 25 nmwere used. The isoelectric point of these bacteriophages is at pH 3.9.15 ml of an aqueous solution of 10⁵ PFU/ml (plaque-forming units/ml)were filtered through the membrane; the pH of the solution was 7.Membranes of polyethersulfone 0.2 μm without a coating and with acoating of titanium (20 nm) and a coating of gold (20 nm) wereinvestigated. The following table shows the results of the experiments:

Coating Voltage in volts Retention in Log 10 without 0.0 0.1 Titanium0.0 0.05 Titanium 1.0 1.4 Gold 0.0 <0.01 Gold 1.0 2.4

It is apparent from the results of the retentionexperiments—particularly for adsorption, without a coating, and withoutvoltage—that a low level of virus retention is achieved on the membranewithout a coating. Polymer membranes have a zeta potential (surfacetension) without a coating. Titanium and gold have a lower zetapotential (near zero). So it is understandable that without voltage onthe membrane without a coating, the most viruses are adsorbed incomparison to the coated membranes, since a gold or titanium coating ofthe membrane reduces the zeta potential and thus the charge of themembrane.

Considering the results of the tests for adsorption under a voltage of1.0 volts, virus retention on the order of 1.4 log₁₀ (i.e., a retentionof >95%) is achieved with the titanium coating, and virus retention onthe order of 2.4 log₁₀ (i.e., >99% retention) is achieved with a goldcoating. It was able to be shown that, by means of a membrane that isprovided with a metal layer and an applied voltage of 0.5 volts or 1.0volts, a significant improvement of the retention, particularlyadsorption, of endotoxins as well as of viruses can be achieved incomparison to an uncharged membrane.

In addition, after switching the voltage in the experiments for theretention of endotoxins from +500 mV to −500 mV, at least 50% of theadsorptively bound endotoxins were able to be recovered back into thewater by rinsing the membrane with water. This demonstrates that atleast some of the endotoxins are desorbed by reversing the voltage.

The object is also achieved by a method for determining the occupancy ofthe binding sites of the polymer membrane with a flat and porous metalcoating and/or for determining at least one at least relativeconcentration in the liquid, this method building on the method for theat least temporary separation and/or detection of charged biologicallyactive substances in a liquid and can make use of all advantageousembodiments and is characterized in that the current flow caused by theapplied voltage is detected and/or evaluated, particularly evaluatedwith regard to falling below a limit and/or exceeding a positiveand/negative rate of change and/or its time course, and an alarm istriggered particularly in the case of an undershoot or overshoot. Thisenables concentration limits or rates of change of concentrations to bemonitored, for example.

Such monitoring can be performed very closely and even in real time. Forinstance, it is possible to monitor the endotoxin concentration in theblood. The current flow is dependent on the occupancy of the bindingsites on the membrane. If fewer binding sites are available, the currentflow drops. The concentration need not be determined as an absolutevalue, however; it is also sufficient to detect under- or overshootingof reference values, and the reference values can also be indicated inthe form of current flows.

For instance, an alarm can occur when certain concentration limitsand/or rates of change are exceeded, particularly measured as the rateof change of the current flow, e.g., acoustically, optically, and/orelectrically.

The object is achieved by electrosorption and/or electrofiltrationdevice comprising a counterelectrode and a polymer membrane with a flatand porous metal coating on at least one side of the polymer membrane,particularly as described above, and a contact of the metal coating forthe purpose of applying a voltage to the counterelectrode. The aboveapplies here to the device as well, particularly with regard toelectrode, membrane, coating, counterelectrode, reference electrode,and/or arrangement.

The polymer membrane and, in particular, the counterelectrode as wellare arranged in a housing that is embodied particularly as a syringeattachment and/or has a small hold-up volume, particularly of no morethan 10 ml and/or no more than 20 mm³/mm² of metal coating on thepolymer membrane, particularly no more than 2 mm³/mm² of metal coatingon the polymer membrane, and/or the polymer membrane and thecounterelectrode are configured to be or are connected to a voltagesource that is set up to form a voltage between the polymer membrane andthe counterelectrode, the voltage source being arranged particularly ina common housing with the polymer membrane and the counterelectrodeand/or a current-measuring device being provided that measures thecurrent flowing between the polymer membrane and the counterelectrodeand/or measures its rate of change and/or compares it with a limitvalue, and/or the voltage source being configured to reverse thepolarity of the voltage.

For the purpose of connecting to a voltage source and/or contacting themetal coating, the metal coating of the membrane can, for example, beconnected directly to and/or brought into contact with an electricalconductor, e.g., a cable, for example by soldering or welding. Inaddition or alternatively, however, it can also be brought into contactwith other electrically conductive components—e.g., with a cage intowhich the membrane is inserted and/or with at least one conductivecoated folding aid—and connected to the voltage source by means of theseand, optionally, additional components and/or cables and/or contacted bysame.

It is also possible to incorporate an inductive and/or capacitivecoupling into the connection to the voltage source so that it is alsopossible, for example, to connect the voltage source through a closedhousing without corresponding cable feedthroughs.

The common housing can also be an outer housing that encloses an innerhousing in which polymer membrane and counterelectrode are arranged.

In particular, a residence volume for liquid is provided in the housingin which liquid can dwell or through which liquid can be moved. The flatand porous metal coating of the polymer membrane and, in particular, thecounterelectrode are then arranged within this volume. This residencevolume is arranged particularly in liquid-permeable connection with atleast one, particularly two connections for hoses and or syringes,particularly in the form of a Luer-lock connection. In particular, thisresidence volume together with the volume of the at least one,particularly two connections and the liquid-permeable connectionrepresents the hold-up volume.

The device can also include a device for generating a voltage, e.g., abattery, particularly in the common housing, that is particularlyarranged and/or contacted such that it can generate a potential betweenmetal coating of the polymer membrane and counterelectrode.

The device is embodied particularly as a pre-filter and/or syringeattachment.

In particular, counterelectrode and polymer membrane with a flat andporous metal coating are electrically insulated from one another.

Electrosorption and/or electrofiltration device particularly has avessel and/or housing for the purpose of arranging and conducting liquidthrough as well, the polymer membrane and the counterelectrode beingarranged so as to be electrically insulated from one another.

The electrosorption and/or electrofiltration device can include one ormore reference electrodes.

It can also advantageously contain at least one additional electrode asdescribed above in relation to the electrosorption and/orelectrofiltration process.

The other features described in relation to this method can also beadvantageously implemented in the electrosorption and/orelectrofiltration device.

The polymer membrane with metal coating is advantageously a metal-coatedpolymer membrane as described above. These are especially suitable,particularly if the porous passages are also coated with metal. Sincethe electrically active surface is then substantially larger. However,other metal-coated polymer membranes can also be used.

To special advantage, the counterelectrode is either an additional flat,porous metal coating on a second side that is situated opposite thefirst side or a permeable electrode that is formed particularly by ametallic mesh with interposition of an insulating and permeable spacer.

In particular, the device for carrying out the method for the at leasttemporary separation and/or detection of charged biologically activesubstances is advantageously configured with some or all advantageousfeatures. In particular, it is also configured to carry out the methodfor determining the occupancy of the binding sites of the polymermembrane with a flat and porous metal coating and/or for determining atleast one concentration in the liquid. The method is carried outparticularly with a device according to the invention.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Additional advantageous embodiments are to be explained below purely forthe sake of example with reference to the schematic drawing thatfollows. In the drawing:

FIG. 1 shows an electrofiltration device according to the invention;

FIG. 2 shows an electrofiltration device according to the invention as asyringe attachment filter; and

FIG. 3 an electrofiltration device according to the invention with afolded membrane that is embodied as a cartridge in a housing.

DETAILED DESCRIPTION

FIG. 1 shows an electrofiltration device according to the invention. Theelectrofiltration method according to the invention can be carried outwith this.

A liquid is introduced into the receiving vessel (5) and filtered intothe collecting vessel (3) through the membrane (6), which is formed by ametal-coated polymer membrane, with application of a voltage, applied bya potentiostat (4), between the membrane (6) that is embodied as anelectrode, and counterelectrode (2). The frit (7) serves the purpose ofstabilizing the membrane.

FIG. 2 shows an electrofiltration device according to the invention inthe form of a syringe attachment filter. Shown is a circular membrane(8) made of polymer material and metallized on one or both sides. Themetallization can be applied to one side, upstream from the membrane(top of the figure) or downstream from the membrane (bottom of thefigure), or to both sides. In the case of application to both sides, themetallized sides are particularly insulated from one another.

In principle, FIG. 2 shows two possible embodiments in one figure.Either all parts that are designated by the suffix b are omitted, or allparts that are designated by the suffix a.

In particular, the filter also has at least one counterelectrode (10 aor 10 b, 12 a or 12 b). This can be embodied as a metal mesh, forexample.

Electrically insulating but permeable release films (9 a or 9 b) thatallow the flow of a liquid in the direction indicated by the arrows, butprovide electrical insulation between the circular membrane (8) andcounterelectrode (10 a or 10 b) can be included. However, an embodimentwithout such a dividing line is also possible if insulation is ensuredby means of other constructive measures.

The syringe attachment filter has a filter inlet (13). It also has afilter outlet (14). These can include feedthroughs for contacting. Thesecan be achieved by cable but also inductively, capacitively, by means ofplugs, and/or the like.

Electrical conductors (12 a or 12 b and 11 a or 11 b) are also included.The conductors (12 a or 12 b) themselves can also serve ascounterelectrodes and thus replace the counterelectrodes (10 a or 10 b).

Filter inlet and filter outlet are particularly part of a housing forthe hermetic sealing of the entire filter, so that a loss of liquid isonly possible between the filter inlet and filter outlet and only bypassing through the elements that are arranged in the liquid flow, suchas circular membrane, trend film, and counterelectrode, insofar as theyare embodied as a permeable or mesh.

The syringe attachment filter is embodied so as to enable a potential tobe applied between metallization of the circular membrane andcounterelectrode. For this purpose, it particularly has correspondingconductors, feedthroughs, and/or contacting and/or transmission devices(in particular inductive and/or capacitive). The voltage can be applied,for example, via a voltage source (15 a or 15 b) and electricalconductors (12 a or 12 b and 11 a or 11 b).

With the aid of such a syringe attachment filter with a voltage source(15 a or 15 b), substances contained in the liquid, such as endotoxins,can be adsorbed on the circular membrane (8) under a first polarizationof the voltage source when liquid, particularly blood, is passed throughthe syringe attachment filter.

Later, the substances can be discharged, particularly under reversepolarization or without voltage and with the passage of another liquid,e.g., water, particularly counter to the direction of flow shown by thearrows.

FIG. 3 shows a cartridge according to the invention in a housing with afolded membrane (16). According to the prior art, such fluted filtercartridges are prepared as follows:

-   -   1. The membrane (16) is positioned and folded between two        folding aids (17 a and 17 b), which are particularly made of a        polymer fleece and/or film.    -   2. The finished folded membrane is pressed into a round cage        (18) so that it does come unfolded again and is provided with a        core. Cage and core are particularly made of plastic and, in        particular, configured such that liquid can pass through,        particularly provided with rectangular or round holes.    -   3. The membrane with folding aid and cage and core are welded to        the upper closure of a sealed plastic cap and then welded to the        lower closure (plate with opening).

The same procedure was also used in this exemplary embodiment and cangenerally be used with the membrane that is included and/or usedaccording to the invention.

FIG. 3 shows a circular membrane (16) made of polymer material andmetallized on one or both sides. The metallization can be applied to oneside, upstream from the membrane (on the outside in the figure) ordownstream from the membrane (on the inside in the figure), or to bothsides. If it is applied to both sides, the metallized sides areparticularly insulated from one another by the membrane itself. Themembrane is folded particularly by means of folding aids that are foldedwith the membrane and consist particularly of a polymer fleece (17 a,b). The polymer fleece can be embodied both as a conductive fleece (withmetal coating on both sides) or as a voltage-insulating fleece.

In principle, FIG. 3 shows two possible embodiments in one figure.Either all parts that are designated by the suffix b are omitted, or allparts that are designated by the suffix a. In the variant in which theparts that are denoted with the suffix a are included, the membrane iscontacted directly via an electrical line as an electrode, or in thevariant in which the parts that are denoted with the suffix b areincluded, the membrane is contacted as an electrode via an electricallyconductive folding aid (17 b) and via an electrically conductive corethat holds the folded membrane.

In particular, the filter also has at least one counterelectrode (25 aor 25 b). This can be embodied as a metal mesh and/or rod electrode, forexample.

The filter that is embodied as a cartridge with folded membrane has afilter inlet. It also has a filter outlet (23). These can includefeedthroughs for contacting. These can be achieved by cable but alsoinductively, capacitively, by means of plugs, and/or the like.

Electrical conductors (24 a and 25 a or 24 b and 25 b) are alsoincluded. The conductors (25 a or 25 b) can serve as counterelectrodesand thus replace the counterelectrodes.

Filter inlet and filter outlet are particularly part of a housing forthe hermetic sealing of the entire diaphragm so that a loss of liquid isonly possible between the filter inlet and filter outlet and only bypassing through the elements that are arranged in the liquid flow, suchas membrane and counterelectrode, insofar as they are embodied as apermeable or mesh.

The cartridge with folded membrane is embodied so as to enable apotential to be applied between metallization of the membrane andcounterelectrode. For this purpose, it particularly has correspondingconductors, feedthroughs, and/or contacting and/or transmission devices(in particular inductive and/or capacitive). The voltage can be applied,for example, via a voltage source (26 a or 26 b) and electricalconductors (25 a or 25 b and 24 a or 24 b).

With the aid of such a cartridge with folded membrane with a voltagesource (26 a or 26 b), substances contained in the liquid can beadsorbed on the folded membrane (8), for example under an initialpolarization of the voltage source upon passage of liquid through thecartridge with folded membrane.

Later, the substances that were previously bound electrosorptively tothe folded membrane of the filter can be desorbed, particularly underreverse polarization or without voltage and, in particular, with thepassage of another liquid, e.g., water, particularly counter to thedirection of flow indicated by the arrows.

LIST OF REFERENCE SYMBOLS

-   -   1 cable    -   2 counterelectrode    -   3 collecting vessel    -   4 potentiostat    -   5 receiving vessel    -   6 diaphragm    -   7 frit    -   8 circular membrane (polymer) with metallization on one side        (upstream or downstream) or on both sides    -   9 a and b electrically insulating, permeable release film    -   10 a and b counterelectrode upstream (a) and downstream (b) from        the switchable circular membrane 1 in the form of a permeable        metal mesh    -   11 a metal wire for contacting of the upstream-side        metallization of the circular membrane 8    -   b metal wire for contacting of the downstream-side metallization        of the circular membrane 8    -   12 a and b electrical conductor to the counterelectrode/metal        mesh 10 a and 10 b    -   13 filter inlet with feedthrough(s) for electrical contacting of        electrode/membrane and counterelectrode    -   14 filter outlet, optionally with feedthrough for electrical        contacting of the counterelectrode/metal mesh    -   15 a voltage source for applying electrical potential to        electrode and counterelectrode    -   b voltage source for applying electrical potential to electrode        and counterelectrode    -   16 folded membrane    -   17 a and b electrically insulating, permeable release film in        front of or behind the membrane    -   18 conductive outer sleeve of the cartridge    -   19 conductive inner sleeve of the cartridge    -   20 upper closure member and baffle plate of the cartridge    -   21 lower closure member and outlet of the cartridge    -   22 22 filter inlet and filter housing    -   23 filter outlet and filter housing    -   24 a contact bushing for electrical contacting of the upstream        metallization of the membrane 16    -   b contact bushing for electrical contacting of the downstream        metallization of the membrane 16    -   25 a contact bushing and counterelectrode on the upstream side        of the metallized membrane 16    -   b contact bushing for electrical contacting of the        counterelectrode    -   c counterelectrode on the downstream side of the metallized        membrane 16    -   26 a voltage source for applying electrical potential to        electrode and counterelectrode    -   b voltage source for applying electrical potential to electrode        and counterelectrode

What is claimed:
 1. A method for the at least temporary separation and/or detection of charged biologically active substances in a liquid by means of electrosorption and/or electrofiltration, comprising the following steps: a. providing a polymer membrane with a flat and porous metal coating at least on a first side of the polymer membrane; b. providing a counterelectrode; c. applying a voltage between the metal coating of the polymer membrane and the counterelectrode; d. bringing the polymer membrane and the counterelectrode into contact with the liquid, with the contacting being performed such that the liquid generates at least one connection between the polymer membrane and the counterelectrode.
 2. The method as set forth in claim 1, wherein step c takes place after steps a and b and before step d, or wherein step d takes place after steps a and b and before step c.
 3. The method as set forth in claim 1, wherein, after steps a to d, the liquid is removed at least partially and/or the liquid is allowed to pass through the membrane at least partially.
 4. The method as set forth in claim 1, wherein, the rinsing of the membrane is performed before or after the polarity reversal or reduction in the voltage.
 5. The method as set forth in claim 1, wherein the polymer membrane with a flat and porous metal coating and the counterelectrode are accommodated in a housing, which has a small hold-up volume of no more than 10 ml and the liquid to be filtered is conducted through the housing.
 6. The method as set forth in claim 5, wherein the housing has a small hold-up volume of no more than 10 ml and no more than 2 mm³/mm² of metal coating on the polymer membrane.
 7. The method as set forth in claim 5 wherein the liquid is pressed out of a syringe and through the housing through actuation of the syringe.
 8. The method as set forth in claim 1, wherein the counterelectrode is formed either by an additional flat, porous metal coating on a second side that is situated opposite the first side, the two-dimensional metal coatings being isolated from each other by the polymer membrane, or through arrangement of a permeable electrode.
 9. The method as set forth in claim 8, wherein the permeable electrode is formed by a metallic mesh with interposition of an insulating and permeable spacer.
 10. The method as set forth in claim 1, wherein the porosity of the polymer membrane with metal coating is reduced by between 1% and 50% relative to the initial bubble point pore and/or the mean pore size compared to the uncoated polymer membrane.
 11. The method as set forth in claim 10, wherein the porosity of the polymer membrane with metal coating is reduced by between 1% and 20% relative to the initial bubble point pore and/or the mean pore size compared to the uncoated polymer membrane.
 12. The method as set forth in claim 1, wherein the thickness of the metal coating is from 5 to 50 nm and the pore size of the uncoated polymer membrane is greater than 0.01 μm.
 13. The method as set forth in claim 1, for determining the occupancy of the binding sites of the polymer membrane with a flat and porous metal coating or for determining at least one concentration in the liquid, wherein the current flow caused by the applied voltage is detected or evaluated, and an alarm is triggered in the case of an undershoot or an overshoot.
 14. The method as set forth in claim 13, wherein the current flow caused by the applied voltage is evaluated with regard to falling below a limit or exceeding a positive or negative rate of change. 